Journal: iScience

Article Title: A rat liver cell atlas reveals intrahepatic myeloid heterogeneity

doi: 10.1016/j.isci.2023.108213

Figure Lengend Snippet: Comparisons between transcriptomic platforms and immunohistochemistry suggest zonation patterns of selected hepatic populations To provide information on the zonation of hepatic populations within hepatic lobules, key cluster markers of each cell type were examined in PC1 and PC2 of each spatial sample. (A) Dot plot indicating the relative expression of marker genes in each population of the snRNA-seq map. The size of the circle indicates the percentage of cells expressing the marker of interest, and the color represents the average expression value of the marker. Expression values of (B) mesenchymal marker Ecm1 (C) cholangiocyte marker Anxa4 (D) myeloid marker Cd163 (E) non-inflammatory myeloid marker Marco (F) myeloid marker Cd68 . Red and dark blue indicate higher and lower expression values in each spot, respectively. (G) Representative spatial distributions of CD68 + cells in the rat liver lobule. Rectangular layers 350 um wide were drawn from the portal tract (layer 1) to the central vein (layer 10) region. Digital images were scanned at 20× magnification. The scale bar represents 100 μm in the full image and 20 μm in the enhanced area. Each rectangular layer is referred to as a region of interest (ROI). (H) Quantification of CD68 + cell densities (#CD68+ cells/layer mm2) in the liver lobule for DA and LEW rats. 30 ROIs were assessed per strain across three animals. A higher number of CD68 + cells were detected near the periportal area. No significant strain-specific differences in the spatial distribution of CD68 + cells were noted. (I) Representative spatial distributions of CD163+ cells in the rat liver lobule. (J) Quantification of CD163+ cell densities (#CD163+ cells/layer mm2) in the liver lobule for DA and LEW rats. The statistical analysis reflects similar strain and zonation patterns as CD68. Statistical significance was determined using a two-way ANOVA followed by Sidak’s multiple comparisons test. (∗: p value <0.05, ∗∗: p value <0.01, ∗∗∗: p value <0.001,∗∗∗∗: p value <0.0001). Data are represented as mean ± SEM. Each dot in H and J represents an ROI region (n = 30). ROI: region of interest, BD: bile duct, CV: central vein, PV: portal vein.

Article Snippet: The resulting NPC was stained with a FITC conjugated anti-rat CD68 recombinant antibody (Miltenyi, Clone:REA237).

Techniques: Immunohistochemistry, Expressing, Marker

Journal: iScience

Article Title: A rat liver cell atlas reveals intrahepatic myeloid heterogeneity

doi: 10.1016/j.isci.2023.108213

Figure Lengend Snippet: Varimax PCs capture rat hepatic cell identity signatures and strain-specific differences (A) Bar plot representing the feature importance scores (mean decrease Gini impurity) of the top 20 features (varimax factors) of the random forest model trained to predict the strain attributes of the rat hepatic cells. Varimax PC5 and 15 are the most informative features to differentiate cells of each strain from another, which indicates the two factors have captured strain-related variations within the map. (B) A correlation heatmap between the average gene expression of each cluster and the loading scores of varimax factors (capturing the contribution of all genes to a factor). Columns are varimax factors and rows are cell populations. Each cell-type cluster is defined by key marker genes, and dark red or blue indicates that the expression of a marker gene set is positively or negatively correlated, respectively, with a particular varimax factor. A high absolute correlation value indicates a match between a varimax factor and a cell-type cluster. (C) The projection of cells over varimax-1 and 5 indicates that the cells from each strain form distinct clusters over varimax-5. (D) Boxplot indicating the distribution of varimax-5 score over each strain. Cells from DA and LEW strains represent significantly different varimax-5 scores (Wilcoxon-test p value <2.2e-16), indicating that varimax-5 has captured strain differences. (E) The top 10 genes on the top (left table) and bottom (right table) of the varimax-5 loading list mainly contain known hepatocyte markers, indicating that varimax-5 has captured hepatocyte-specific strain differences. Genes with high positive scores (left table) are associated with the DA strain and genes indicating negative loading scores (right table) are LEW-related. The absolute loading scores indicate the contribution of each gene to the corresponding factor. (F) Projection of cells over varimax-1 and 15 indicates that a population of cells from each strain (dotted lines) forms distinct clusters over varimax-15. Annotation of the selected cells indicates that they are mainly from the Marco + myeloid cluster 5. (G) Boxplot indicating the distribution of hepatic cells based on strain over varimax-15. (Wilcoxon-test p value <2.2e-16). The outlier data points (dotted lines) are mainly myeloid cells. (H) The top 10 genes with positive (right table) and negative (left table) varimax-15 loading scores are immune-response related. Genes with positive scores (right table) are associated with the LEW strain, and genes indicating negative loading values (left table) are DA related. The absolute loading scores indicate the contribution of each gene to the corresponding factor. (I) Expression pattern of known myeloid marker genes Marco , Vsig4 , Cd68 , and Lyz2 over UMAP. Dark green represents high expression values. The distribution of general myeloid markers ( Cd68 , Vsig4 ) and non-inflammatory myeloid marker ( Marco ) is consistent with the varimax-15 distribution ( Figure 2 J). (J) The UMAP projection of cells colored based on the varimax-15 score shows the enrichment of varimax-15 over Marco + myeloid population (cluster 5). Darker colors represent higher values of varimax-15 scores. Data are represented as mean ± SEM with each dot representing a single cell. Corrcoef.: correlation coefficient, Var: varimax PC. varimax PCs are referred to as PCs within the main text.

Article Snippet: The resulting NPC was stained with a FITC conjugated anti-rat CD68 recombinant antibody (Miltenyi, Clone:REA237).

Techniques: Gene Expression, Marker, Expressing

Journal: iScience

Article Title: A rat liver cell atlas reveals intrahepatic myeloid heterogeneity

doi: 10.1016/j.isci.2023.108213

Figure Lengend Snippet: The inflammatory potential of myeloid cells found in LEW rats is greater than that found in DA rats Myeloid cell inflammatory potential was evaluated after lipopolysaccharide (LPS) stimulation of freshly isolated liver-resident non-parenchymal cells. LPS-induced TNFα secretion was measured via intracellular cytokine staining (ICS). The non-parenchymal liver cell dissociate was obtained via a gentle enzymatic perfusion process and differential centrifugation. The resulting cells were plated in 12 well plates for 3.5 h before being stimulated for 6 h under a concentration of 1 ng/mL of LPS in the presence of 1:1000 concentration of Monensin and Brefeldin. (A) Flow cytometry plots showing the gating strategy for macrophages. (B) Percentage of TNFα + secreting CD68 + CD11b + myeloid cells in the unstimulated control and stimulated conditions of Dark Agouti and Lewis macrophages. (C) Summary graphs of Lewis versus Dark Agouti total TNFα as a percentage of CD68 + CD11b + myeloid, (D) and of the mean fluorescence intensity (MFI) of Lewis vs. Dark Agouti TNFα. (E) Representative flow cytometry plot of TNFα secretion patterns based on ITGAL subpopulations. (F) and summary graph ITGAL expressing CD68 + CD11b + myeloid subpopulations. Plotted are the values from all 4 experimental replicates. Statistical significance for ICS was determined using a non-parametric 2 tailed Mann-Whitney test. (n = 4) (G) Cytometric bead array (LEGENDplex) was performed to quantify the level of cytokines (TNFα, Il-18, CXCL1) on culture supernatants of enriched CD68 + myeloid cells after 24 h of stimulation in various LPS concentration conditions (0, 0.05, 0.1, 1, 10 ng/mL). Three technical replicates were used per animal. Statistical significance of the CBA was determined using a two-way ANOVA and Sidak’s multiple comparisons test (n = 3) Data are represented as mean ± SEM with each dot representing a single animal. (∗: p value <0.05, ∗∗: p value <0.01, ∗∗∗: p value <0.001,∗∗∗∗: p value <0.0001) DA: dark agouti, LEW: lewis, SSC-A: side scatter area, FSC-A: forward scatter area.

Article Snippet: The resulting NPC was stained with a FITC conjugated anti-rat CD68 recombinant antibody (Miltenyi, Clone:REA237).

Techniques: Isolation, Staining, Gentle, Centrifugation, Concentration Assay, Flow Cytometry, Control, Fluorescence, Expressing, MANN-WHITNEY

Journal: iScience

Article Title: A rat liver cell atlas reveals intrahepatic myeloid heterogeneity

doi: 10.1016/j.isci.2023.108213

Figure Lengend Snippet:

Article Snippet: The resulting NPC was stained with a FITC conjugated anti-rat CD68 recombinant antibody (Miltenyi, Clone:REA237).

Techniques: Recombinant, SYBR Green Assay, Staining, Selection, Gene Expression, Software